Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Science, Wuhan 430064, China.
BMC Biotechnol. 2012 Jul 6;12:39. doi: 10.1186/1472-6750-12-39.
Widely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired.
This paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolecular homologous recombination in bacteria for native DNA cloning. ABI-REC was developed to precisely clone inserts into defined location in a directional manner within recipient plasmids. It featured an asymmetric 3-primer PCR performed in a single tube that could robustly amplify a chimeric insert-plasmid DNA sequence with homologous arms at both ends. Intramolecular homologous recombination occurred to the chimera when it was transformed into E.coli and produced the desired recombinant plasmids with high efficiency and fidelity. It is rapid, and does not involve any operational nucleotides. We proved the reliability of ABI-REC using a double-resistance reporter assay, and investigated the effects of homology and insert length upon its efficiency. We found that 15 bp homology was sufficient to initiate recombination, while 25 bp homology had the highest cloning efficiency. Inserts up to 4 kb in size could be cloned by this method. The utility and advantages of ABI-REC were demonstrated through a series of pig myostatin (MSTN) promoter and terminator reporter plasmids, whose transcriptional activity was assessed in mammalian cells. We finally used ABI-REC to construct a pig MSTN promoter-terminator cassette reporter and showed that it could work coordinately to express EGFP.
ABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design. ABI-REC is a novel approach to DNA engineering and gene functional analysis.
广泛使用的依赖于限制酶的克隆方法既费时又费力，而几种类型的连接酶独立的克隆方法具有固有局限性。人们非常希望有一种快速可靠的方法将天然 DNA 序列克隆到所需的质粒中。
本文介绍了 ABI-REC，这是一种将不对称桥 PCR 与细菌内分子同源重组相结合的新型策略，用于天然 DNA 克隆。ABI-REC 的开发目的是精确地将插入物以定向方式克隆到受体质粒中的指定位置。它的特点是在单个管中进行不对称的 3 引物 PCR，可以有效地扩增带有同源臂的嵌合插入质粒 DNA 序列。当它转化为大肠杆菌时，分子内同源重组发生，产生具有高效率和保真度的所需重组质粒。它快速，不涉及任何操作核苷酸。我们使用双抗性报告基因检测来证明 ABI-REC 的可靠性，并研究了同源性和插入物长度对其效率的影响。我们发现 15 个碱基对的同源性足以启动重组，而 25 个碱基对的同源性具有最高的克隆效率。通过这种方法可以克隆大小达 4 kb 的插入物。ABI-REC 的用途和优点通过一系列猪肌肉生长抑制素（MSTN）启动子和终止子报告质粒得到了证明，其在哺乳动物细胞中的转录活性得到了评估。我们最后使用 ABI-REC 构建了猪 MSTN 启动子终止子盒报告，并证明它可以协同工作以表达 EGFP。
ABI-REC 具有以下优点：（i）快速高效；（ii）不引入额外碱基的天然 DNA 克隆；（iii）无限制酶；（iv）由于设计了特定的引物，易于定向和定点重组。ABI-REC 是一种新的 DNA 工程和基因功能分析方法。
